ARF is a recently characterized GTP-binding regulatory protein component of the adenylate cyclase system. These studies are aimed at the elucidation of the role of ARF in signal transduction, differentiation, and proliferation. We have used the bovine ARF gene to clone two homologous genes, ARFI and ARF2, from the yeast, S. Cerevisiae. Disruption of ARF1 leads to slow growth, cold sensitivity, and supersensitivity to fluoride. We plan to investigate the consequences of ARF2 disruption both alone and in combination with disruption of ARF1. Suppressors of the arf1- phenotype have been obtained and will be analyzed. It is expected that some of these suppressor genes will code for other proteins in the ARF pathway. We will determine the ability of human ARF to rescue the arf1- phenotype to test for conservation f ARF function. In addition, we are cloning and sequencing ARF from both human and Xenoous laevis libraries. The human gene will be used to screen a variety of human tumors for the presence of abnormal ARF expression. The Xenopus gene will be used in studies of ARF function in Xenopus oocytes which allow microinjection of purified ARF proteins and biochemical assays of resultant changes in cellular metabolism.